Modified AdCTLA-4 vector blocks alloimmune response in vitro

Background. Gene transfer of the costimulatory blockade molecule CTLA-4Ig i nto cold-preserved rat liver allografts results in indefinite allograft sur vival. Despite local delivery, this mode of immunomodulation is associated with systemic immunosuppression. In an effort to restrict immunosuppression to the graft, we have constructed a novel adenoviral vector, AdCTLA-4ex-TA G;, in which the Ig sequence of CTLA-4Ig DNA has been deleted to destabiliz e the gene product to promote rapid extrahepatic degradation while maintain ing its immunosuppressive activity within the graft milieu. Methods, (1) Vector construction. CTLA-4 extracellular binding domain (CTLA -4ex) was prepared by PCR-based cloning methods and fused in frame to a gen etic element encoding an epitope TAG;allowing identification of the transge ne product CTLA-4exTAG. CTLA-4exTAG was subcloned into a shuttle vector ena bling isolation of AdCTLA-4exTAG. (2) In vitro transfection: AdCTLA-4exTAG was transfected into MH1C1 cells and then supernatant was recovered for Wes tern blot analysis. (3) In vitro alloimmune response was characterized by C FSE proliferation assay. (4) Extrahepatic effect of AdCTLA-4exTAG was chara cterized by the ability to control tumor growth after implantation of a reg ressive, immune sensitive cancer cell line (REGb) in the skin of BDM rats a fter liver transduction with AdCTLA-4exTAG. Results. Expression and secretion of the recombinant protein were documente d by Western blot after infection of the MH1C1 cell line with AdCTLA-4exTAG . Addition of infected MH1C1 cell supernatant resulted in abrogation of all oimmune response as shown by markedly diminished division of CD4(+) T cells in a CFSE proliferation assay. Extrahepatic tumor regressed normally after liver transduction with AdCTLA-4exTAG. Conclusions. These results show efficient in vitro expression of CTLA-4exTA G after transfection with AdCTLA-4exTAG. The modified protein retains its a bility to abrogate in vitro alloimmune response. Efficient control of extra hepatic tumor growth after liver-directed delivery of AdCTLA-4exTAG suggest s that the immunosuppressive effect of this vector is restricted to the liv er. These results set the ground for the utilization of this novel adenovir al vector in the transplant setting. (C) 2001 Academic Press.